Effect of tetrahedral framework nucleic acids on the reconstruction of tendon-to-bone injuries after rotator cuff tears

Abstract

Clinicians and researchers have always faced challenges in performing surgery for rotator cuff tears (RCT) due to the intricate nature of the tendon-bone gradient and the limited long-term effectiveness. At the same time, the occurrence of an inflammatory microenvironment further aggravates tissue damage, which has a negative impact on the regeneration process of mesenchymal stem cells (MSCs) and eventually leads to the production of scar tissue. Tetrahedral framework nucleic acids (tFNAs), novel nanomaterials, have shown great potential in biomedicine due to their strong biocompatibility, excellent cellular internalisation ability, and unparalleled programmability. The objective of this research was to examine if tFNAs have a positive effect on regeneration after RCTs. Experiments conducted in a controlled environment demonstrated that tFNAs hindered the assembly of inflammasomes in macrophages, resulting in a decrease in the release of inflammatory factors. Next, tFNAs were shown to exert a protective effect on the osteogenic and chondrogenic differentiation of bone marrow MSCs under inflammatory conditions. The in vitro results also demonstrated the regulatory effect of tFNAs on tendon-related protein expression levels in tenocytes after inflammatory stimulation. Finally, intra-articular injection of tFNAs into a rat RCT model showed that tFNAs improved tendon-to-bone healing, suggesting that tFNAs may be promising tendon-to-bone protective agents for the treatment of RCTs.

FIGURE 1

The use of tFNAs improves the healing process of tendon‐to‐bone injuries following rotator cuff tears.

FIGURE 2

Production and characterisation of tFNAs. (A) S The process of creating tFNAs is illustrated in a schematic diagram. (B) Size distribution of tFNAs. (C) Zeta potential distribution. (D) AFM image. (E) PAGE indicating the molecular weight. (F) The surface morphology and average size observed by TEM imaging.

FIGURE 3

Effect of tFNAs on inflammasome assembly in RAW 264.7 cells under inflammatory conditions. (A) Cellular uptake of ssDNAs and tFNAs. (B) Gene expression of iNOS, CD206, IL‐1β, and IL‐6 in RAW264.7 cells treated with LPS and tFNAs. Data are presented as the mean ± SD (n = 3). (C) Protein expression of ACS, NLRP3, pro‐caspase‐1, caspase‐1, and IL‐1β in RAW 264.7 cells treated with LPS and tFNAs. (D) Quantitative analysis of the protein expression levels of ACS, NLRP3, pro‐caspase‐1, caspase‐1 and IL‐1β. Data are presented as the mean ± SD (n = 3). (E) Schematic diagram of the inhibition of inflammasome activation by tFNAs. Statistical analysis: *p < 0.05, **p < 0.01.

FIGURE 4

Under inflammatory circumstances, tFNAs enhance the osteogenic differentiation of BMSCs. (A) Schematic diagram of osteogenic differentiation induction in BMSCs treated with LPS and tFNAs. (B) Alizarin red staining was used to detect the osteogenic differentiation of BMSCs following a 14‐day treatment with LPS and tFNAs. (C) Gene expression of ALP, OPN, and RUNX2 in BMSCs treated with LPS and tFNAs for 1 day. (D) WB analysis was performed to examine the levels of RUNX2 and OPN expression in BMSCs after treatment with LPS and tFNAs for a duration of 3 days. (E) Analysing the levels of protein expression for RUNX2 and OPN in BMSCs that were exposed to LPS and tFNAs using quantitative methods. The data are displayed as the average plus standard deviation (n = 3). (F), (G). Immunofluorescence detection of RUNX2 and OPN. (Green F‐actin, blue nucleus, and red protein.) The measurement bars have a length of 50 μm. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

tFNAs enhance the chondrogenic differentiation of BMSCs under inflammatory conditions. (A) H&E, Alcian blue and safranin‐O staining and immunohistochemical analysis of Col II in pellets treated with LPS and tFNAs for 21 days. (B) Gene expression of SOX‐9, Aggrecan, Col II, and Col I (n = 3). (C) Analysis of the expression levels of SOX‐9, Aggrecan, and Col II in pellets was conducted by WB. (D) quantitative assessment was conducted on the protein expression levels (n = 3). Statistical analysis: *p < 0.05, **p < 0.01.

FIGURE 6

tFNAs regulate tenocyte protein expression under inflammatory conditions. (A) Schematic illustration of the regulation of collagen expression by tFNAs in tenocytes treated with LPS and tFNAs. (B) Gene expression of Col I and Col III in tenocytes treated with LPS and tFNAs for 1 day. (C) WB analysis of the expression levels of Col I, Col III, and TNMD in tenocytes treated with LPS and tFNAs for 3 days. (D) Quantitative analysis of the protein expression levels of Col I, Col III, and TNMD in tenocytes treated with LPS and tFNAs (n = 3). (E), (F) Detection of Col I and Col III using immunofluorescence (Green F‐actin, blue nucleus, and red protein). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

In vivo experimental design and imaging and biomechanical evaluation. (A), (B) Schematic diagram of the rat RCT model. (C) Schematic diagram of the time of intra‐articular injection of tFNAs and the precise time point of sacrifice of experimental rats after the operation. (D) MRI images showing a new tendon in the repaired tissue. (E) Micro‐CT images revealing bone regeneration. (F), (G) Comparative assessment of BMD and BV/TV in different groups (n = 4). (H), (I). The maximum load and stiffness values based on the force–displacement curve (n = 3). Statistical analysis: *p < 0.05, **p < 0.01.

FIGURE 8

Histological analysis in vivo. (A), (B) At 6 weeks and 12 weeks, the regenerated area was examined using histological techniques including H&E, safranin‐O, Masson, and Sirius red staining, as well as immunohistochemical analyses of collagen I and collagen III. (B: bone; I: interface; T: tendon). (B) Histological scores at the tendon‐bone insertion site after 6 and 12 weeks. (C) Tendon maturation scores in the different groups at 6 and 12 weeks (n = 3). Statistical analysis: **p < 0.01.